This invention concerns a reagent system for volume differentiation of at least two populations of leukocytes including a blood diluent especially suitable for use in electronic enumeration and sizing of blood cells, determination of hemoglobin and their collective indices and platelet parameters in a single blood cell sample by means of suitable electronic instrumentation, and a stromatolyzing reagent for use therewith.
The diluent comprises a stable water solution of chemical salts providing an electrolytic solution capable of conducting current to which a blood sample can be added so as to dilute the red blood cells, white blood cells, platelets and other blood components and enable the desired parameters of these blood components to be measured, counted and evaluated.
It is a common medical diagnostic procedure to analyze and test a blood sample of a patient in order to make certain classic determinations with respect to the blood sample. This procedure is an important tool for the physician. Six characteristically important parameters are referred to as red blood cell count (RBC), the hematocrit (HCT), the hemoglobin (Hgb), the mean corpuscular volume (MCV), the mean corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin concentration (MCHC). A seventh important determination is white blood cell count (WBC).
Much effort has been devoted to the development of satisfactorily automated leukocyte differential systems. However, a need exists for reagent systems which will be easily adaptable to automatic blood counting instruments. In particular, it is desirable to develop reagents and methods for use with the COULTER COUNTER Model S Plus series of automated blood cell counters, manufactured by Coulter Electronics, Inc. of Hialeah, Fla., which will enable the cell volume data accumulated on a COULTER CHANNELYZER to discriminate: (1) a lymphoid and a myeloid population, as described in U.S. Pat. No. 4,346,018, and (2) a lymphocyte, monocyte and granulocyte population, as described in copending U.S. application Ser. No. 454,926, now U.S. Pat. No. 4,485,175. Such data are useful as a screening tool for spotting abnormal leukocyte ratios. Abnormal situations flagged out by this method give information of diagnostic significance, and for further study. COULTER, COULTER COUNTER and CHANNELYZER are registered trademarks of Coulter Electronics, Inc.
Separation of normal human leukocytes by volume distribution was first documented by Gauthier and colleagues, (Gauthier, J., Harel, P., Belanger, C. and Fraysse, J., Can. Med. Assoc. J. 97, 793, (1967) and Van Dilla and colleagues, (Van Dilla, M. A., Fulwyler, M. J. and Boone, I. U., Proc. Soc. Esp. Biol. Med. 125, 367, in 1967) as a possible clinical diagnostic method utilizing the principle of counting and sizing developed by Wallace H. Coulter and employed in COULTER COUNTER instruments. These methods were based on the fundamental property of all living cells to regulate their cell volume by genetic code information. Each type of cell in the circulating blood has its own characteristic volume ranging from as small as 3 fL for platelets to more than 450 fL for polymorphonuclear cells (fL designates 1.times.10.sup.-15 liters, a femtoliter). Advanced COULTER COUNTER instruments have been designed to make use of this volume differential for the purposes of counting and determining the size distribution of cell components and to detect and monitor pathological states.
Electrical sizing of particles in suspension by a COULTER COUNTER type instrument has been previously described and documented by many clinical hematology investigators. It is well known that the form and size of the electrical pulse generated by a particle passing through a defined electrical field is influenced by several factors including size, shape and conductance of the particles being counted. In blood cell preparations diluted in an isotonic salt solution, conductivity of the cell membrane is far lower than conductivity of the diluent, and therefore, blood cells may be considered to be electrically non-conducting for practical considerations.
Erythrocytes and the lymphoid leukocytes unfortunately overlap considerably in cell size, and it is not possible to count one in the presence of the other by size discrimination alone. Traditional practice involves the use of a strong reagent that stromatolyses the erythrocytes, reducing them to very small particles or causing membrane solubilization, and at the same time strips the cytoplasm from the leukocytes, leaving only the resistant nuclei to be counted. Since original cell volume is drastically affected and reduced to a minimum, only a single population is visible by size distribution analysis.
The COULTER COUNTER Model S Plus automated blood cell counter is designed to dilute a sample of whole blood in an isotonic diluent, add a stromatolysing reagent, and then begin counting after a few seconds. Data typically are collected for a very few seconds for erythrocytes, leukocytes and platelets; however, an increase in count cycles of a few seconds each can be invoked automatically by the instrument system to achieve need statistical accuracy. Thus, a diluent-lysing reagent system for measuring more than one population of leukocytes must provide erythrocyte lysing kinetics sufficiently rapid to effect complete stromatolysation of the erythrocytes during the lysing period. Changes in leukocyte volume must be minimal during the data collection step and ideally should be stable for several minutes. The reagent system must also preserve the integrity of the erythrocyte and platelet number and size distribution, the hemoglobin absorbance curve and the total leukocyte count. Finger stick bloods should be stable when pre-diluted in the isotonic diluent for at least two hours.
To achieve an analysis of the relative populations of lymphoid and myeloid cells in the blood, the leukocyte volume histogram must show cleanly separated lymphoid and myeloid peaks, with little erythrocyte debris, allowing valleys very close to the baseline. Integration of each peak will give the relative populations of the lymphoid and myeloid cells. The lymphoid peak has been demonstrated to contain lymphocytes and variant lymphocytes, while the myeloid peak contains polymorphonuclear cells, bands, monocytes, eosinophils, basophils and other abnormal cells.
In U.S. Pat. No. 3,874,852 (1975) to Coulter Diagnostics, Inc., a reagent formula is included for a composition containing one quaternary ammonium salt detergent and potassium cyanide to be employed as a lysing and chromagen-forming reagent for obtaining a total leukocyte count, without regard for subpopulations, and hemoglobin determination in the COULTER COUNTER Model S. Further investigations was required to use a plurality of quaternary ammonium salts as lysing agents for obtaining the two-population leukocyte count.
In U.S. Pat. No. 4,286,963 (1981) to Coulter Electronics, Inc. a lytic diluent for the rapid lysing of red blood cells in whole blood for making a differential determination of lymphoid/myeloid populations of leukocytes, and also measuring hemoglobin by chromagen formation, contains a mixture of an aqueous saline solution of at least one quaternary ammonium salt having surface acting properties, and certain additives such as 2-phenoxyethanol.
It is known that two volume distribution analysis is difficult because, with many reagent systems, the two populations rapidly move into one so that there is not enough time within which to make the computations for analysis.
In U.S. patent application, Ser. No. 454,926, filed Jan. 3, 1983, now U.S. Pat. No. 4,485,175, a method and reagent system is described for three-volume differential determination of lymphocyte, monocyte and granulocyte populations of leukocytes.
In U.S. patent application, Ser. No. 295,933, filed Aug. 24, 1981, now U.S. Pat. No. 4,384,971, issued May 24, 1983, there is described a cleansing composition for electronic particle counting apparatus, and method for its use. This composition includes a non-hemolytic polyoxyethylated alkylphenol detergent, dimethylolurea and 1-hydroxypyridine-2-thione which is adjusted to a predetermined osmolality.